Protein Crystal Growth (PCG) in the GN2 Dewar (Phase 1A)

Crytsal Morphologies Crystal Morphologies

Objectives

This investigation will grow protein crystals by using the batch and liquid-liquid diffusion methods (precipitant solution into protein solution) coupled with the flash freezing method in the Dewar. The objectives of this experiment were to: (1) grow better quality protein crystals in space, (2) evaluate the effectiveness of different methods of growing protein crystals, (3) determine if large numbers of samples can be processed by using the Dewar, and (4) determine if protein crystals' growth over a long-duration mission were equivalent to short-duration missions.

Shuttle-Mir Missions
Mir 19/STS-74

Approach
PCG-GN2 Dewar investigated two simple, but elegant methodologies for protein crystal growth, namely, batch and liquid-liquid diffusion. Liquid-liquid diffusion samples consisted of an interface between the protein and the precipitant solution. Conversely, batch samples consisted of a protein and precipitant solution that were premixed into a homogenous solution. Samples were prepared prior the mission. Solutions were placed into capillary tubes varying in length; by varying the length of the sample tubes, scientists could control the diffusion rate. Immediately after the solutions were added and the tubes were sealed, the samples were flash frozen in liquid nitrogen. Once frozen, the samples are stable and no crystal formation can proceed until the samples thaw.

The Dewar and samples were carried into space on STS-71, July 27, 1995. They were transferred to the Russian Space Station Mir following the first docking achieved between a US Space Shuttle and the Mir. The Dewar was fixed to an internal partition of the Mir. It remained there undisturbed until the second docking mission of STS-74 with Mir. Docking occurred on November 14, when the Dewar was transferred back to the Space Shuttle. The Dewar was opened on November 21 and the contents were examined and photographed using microscopy with polarized light. Nineteen different proteins were examined during this mission. This included a total of 183 samples.

Results
PCG-Dewar demonstrated the successful approach to the crystallization of biological macromolecules in space over extended time periods. Of the nineteen types of proteins and viruses investigated, seventeen were crystallized during the Phase 1A mission. However, it is believed that the length of the mission (4 months), judging from the amount of precipitate and degradation of some samples, was probably too long, and a shorter duration might have yielded better results.

The distribution of crystals among the three types of containers used indicated small samples yielded results equal or better than larger and that long diffusion path lengths were better. Distribution of crystals within the container tubes showed a striking gradient of quality and size that indicated long, narrow tubes yield superior crystals as was predicted from other similar experiments.

Earth Benefits
Protein crystal growth is often hindered by sedimentation and convection effects caused by gravity. Therefore, microgravity offers an advantage to protein crystal growth resulting in larger and better quality crystals for x-ray diffraction. Improved x-ray diffraction data could lead to a better understanding of the structure and function of proteins, thus, resulting in the development of new pharmaceuticals and disease treatment.

Publications
Koszelak, Stanley, Leja, Cathy, McPherson, Alexander, Crystallization of Biological Macromolecules from Flash Frozen Samples on the Russian Space Station Mir, Biotechnology and Bioengineering, Vol. 52, pp. 449-458 (1996).

Principal Investigators
Stanley Koszelak
University of California at Riverside

Co-Investigators
Cathy LejaK
Alexander McPherson

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